The transcription factor, IRF-8, blocks neutrophil lineage commitment at several stages of hematopoietic stem cell differentiation. Previous findings revealed that Irf8-/- mice as well as humans and mice with select IRF-8 mutations overproduce neutrophils. We found that IRF-8 is necessary and sufficient to block neutrophil differentiation in common myeloid and common lymphoid progenitor cells, CMPs and CLPs, respectively. Gene expression profiles in Irf8-/- CMPs and CLPs compared to controls revealed that distinct target genes were dysregulated in each of these developmental pathways. The proposed research focuses on the molecular mechanisms by which IRF-8 regulates commitment to the neutrophil lineage. IRF-8 must heterodimerize with other transcription factors in order to bind DNA. In Aim 1, we will determine the co-factors necessary for IRF-8 to regulate neutrophil differentiation and determine the direct target genes of these complexes. IRF-8 forms complexes with the transcription factor, PU.1. Interestingly, reduced PU.1 levels also result in increased neutrophil differentiation. We hypothesize that IRF-8/PU.1 complexes regulate the expression of genes that are required to block neutrophil differentiation in CMPs and CLPs. To test this, we will use previously described IRF-8 mutants that lack the ability to bind co-factors and/or DNA. Importantly, some of these mutants recapitulate the neutrophil phenotype observed in Irf8-/- mice while others result in normal neutrophil differentiation. We will analyze the abiliy of these mutants to bind to PU.1 and transactivate known IRF-8/PU.1 target genes. To identify the IRF-8/PU.1 target genes that block neutrophil differentiation in CMPs and CLPs, IRF-8 and PU.1 ChIP-seq profiles will be compared and contrasted between progenitor cells that express wild type IRF-8 or mutant IRF-8. We will determine whether these candidate factors can block neutrophil differentiation in Irf8-/- progenitor cells in vitro. Some genes that are required to blck neutrophil lineage commitment downstream of IRF-8 might not be direct targets of IRF-8 or might not be exclusively regulated by IRF-8/PU.1 complexes. Thus, in Aim 2 we will employ a candidate-based approach to identify genes regulated by IRF-8 in CMPs and CLPs that limit neutrophil differentiation. We have selected candidate transcription factors that were shown to be downregulated in Irf8-/- CMPs or CLPs via microarray. These factors block neutrophil production or decrease the expression of myeloid genes in vitro. We hypothesize that these candidate genes are the functional downstream targets of IRF-8 that block neutrophil differentiation. We will determine whether these factors block the aberrant neutrophil development in Irf8-/- CMPs and CLPs in vitro and in vivo. Very little is known about the molecular mechanisms by which IRF-8 regulates neutrophil differentiation. This data will significantly increase our understanding of neutrophil lineage commitment and could uncover novel therapeutic targets for diseases in which neutrophils are dysregulated, including myelodysplastic syndromes, myeloid leukemias and various neutropenias.